Brown midrib (BMR) and plant age impact fall armyworm (Spodoptera frugiperda) growth and development in sorghum-sudangrass (Sorghum x drummondii)

Economic losses from insect herbivory in agroecosystems has driven the development of integrated pest management strategies that reduce pest incidence and damage; however, traditional chemicals-based control is either being complemented or substituted with sustainable and integrated methods. Major sustainable pest management strategies revolve around improving host plant resistance, and one of these traits of interest is Brown midrib (BMR). Originally developed to increase nutritional value and ease of digestion for animal agriculture, BMR is a recessive plant gene usually found in annual grasses, including sorghum and sorghum-sudangrass hybrids. In sorghum-sudangrass, BMR expressed plants have lower amounts of lignin, which produces a less fibrous, more digestible crop, with possible implications for plant defense against herbivores- an area currently unexplored. Fall Armyworm (FAW; Spodoptera frugiperda) is a ruinous pest posing immense threat for sorghum producers by severely defoliating crops and being present in every plant stage. Using FAW, we tested the effect of seed treatment, BMR, and plant age on FAW growth, development, and plant defense responses in sorghum-sudangrass. Our results show that seed treatment did not affect growth or development, or herbivory. However, presence of BMR significantly reduced pupal mass relative to its non-BMR counterpart, alongside a significant reduction in adult mass. We also found that plant age was a major factor as FAW gained significantly less mass, had longer pupation times, and had lower pupal mass on the oldest plant stage explored, 60-days, compared to younger plants. These findings collectively show that pest management strategies should consider plant age, and that the effects of BMR on plant defenses should also be studied.

performance Sorghum (Sorghum bicolor), is one of the most agriculturally important crops grown across the world, historically being grown for centuries in Africa and Asia 1 .However, sorghum production in the United States has exponentially increased since the 1980s, and the United States is now producing approximately 25% of the world's sorghum crop 1,2 .Similar to other grasses, sorghum and sorghum-sudangrass (Sorghum x drummondii) face attack from over 150 insect species, many of them attacking specific parts of the plant that include, but are not limited to, sorghum shoot fly (Atherigona soccata), multiple stem borers (Chilo partellus, Busseola fusca, Diatraea saccharalis), armyworms (Spodoptera exemtpa and S. frugiperda), aphids (Melanaphis sacchari, Schizaphis graminum), amongst others 3,4 .To combat the large swath of pests that constantly damage sorghum leading to Texas, USA) alongside non-treated control seeds.We used sorghum-sudangrass as our study system since the crop is commonly grown for feed, has been previously used for similar studies 42 , and was commercially available with BMR and non-BMR genotypes.We then designed FAW growth and development bioassays structured around the following questions: (1) does the presence of BMR influence the growth and development of FAW under short exposure (2) does the presence of BMR influence the amount of herbivory on sorghum-sudangrass plants at different plant ages, (3) do commercially available seed treatments influence the growth and development of FAW, and (4) whether FAW exposed to BMR sorghum-sudangrass or seed-treated sorghum-sudangrass are able to pupate and eclose successfully, measured in pupal and adult mass?We hypothesized that BMR will have differential effects on the growth and development of FAW on BMR sorghum-sudangrass but also on herbivory related injury suffered by the plant.We predicted that older sorghum-sudangrass plants will be more detrimental to FAW development, based on a previous study that reported more damage on younger BMR leaves and significantly less damage on older BMR leaves 31 .We also hypothesized that seed-treated sorghum-sudangrass will have little to no effect on the growth and development of FAW, nor will it reduce its herbivory as multiple studies have shown low success of systemic insecticides, especially in later instars 43,44 .

Plant material-sorghum-sudangrass
For all sorghum-sudangrass plant genotypes (S425 -No BMR) treated and untreated, S60 (Contains BMR) treated and untreated, S72 (Contains BMR) treated and untreated, described in this study were obtained from Richardson Seeds (Richardson Seeds Ltd, Vega, Texas, USA).These genotypes were chosen because of their similarities in growth traits.Plants were grown in DL33 Deepot Tree Pots (6.9 cm diameter, 20.3 cm deep, Greenhouse Megastore, Danville, Illinois, USA) with seeds being sown in LB15 potting soil (Farmers Co-Op, Van Buren, Arkansas, USA).The plants were fertilized with Osmocote Plus 15-9-12 (ICL Specialty Fertilizers) every 14 days and received iron chelate micronutrient (Sprint 330 Chelated Iron 10%) every 14 days.Plants were grown in a greenhouse with a 16-h-light/8-h-dark photoperiod, 28 °C, 50-60% relative humidity.The following experiments were replicated on all three of the following sorghum-sudangrass stages, indicated in days after germination: 10-days old (3-leaf stage), 25-days old (panicle inflation stage), 60-days old (booting stage).Voucher specimens for the species have been previously deposited (after identification) at herbarium from previous work on this species.Permissions to conduct experiments and collect seeds have been obtained from the seed company.All experimental protocols followed institutional, national, and international guidelines and legislation.

Insects-fall armyworm
FAW were purchased as eggs (Frontier Agricultural, Newark, Delaware, USA).FAW eggs were allowed to hatch inside the laboratory at 25 °C and were then reared on an artificial wheat-based germ diet (Product Code: F9772; Frontier Agricultural, Newark, Delaware, USA).The diet was made as per specifications from the supplier, as well as our previous work: 1000 mL of water was heated in an iron cooking pot on a hot plate with mechanical stirring until boiling, followed by the addition of 200 g of General-Purpose Lepidoptera Diet added in slowly to be thoroughly mixed without clumping.Once thoroughly mixed, 8 g Agar powder was added into the mixture and mixed thoroughly again.The completed mixture was added to plastic Sterilite 6-quart storage boxes (Walmart; Bentonville, Arkansas, USA) and left at room temperature for 4 hours for cooling before being refrigerated 42 .

Experiments
Snapshot exposure experiment FAW were reared on artificial diet until reaching 2nd instar, at which larvae were then weighed before being placed on sorghum-sudangrass plants in the greenhouse.FAW were placed within bags, and these bags were then securely tied to sorghum-sudangrass plants using drawstring organza bags (10.2 cm × 15.2 cm, Volcanic, Amazon, Seattle, Washington, USA) with one FAW per plant.Fifteen caterpillars were individually placed on each plant per each of the 6 treatments, S425 (non-BMR) treated and untreated, S60 (BMR) treated and untreated, S72 (BMR) treated and untreated (N = 90), alongside 30 caterpillars continuing to be reared on artificial diet as a control (N = 30).FAW fed on plant material within the bag for 48 h before being removed from the plant and weighed again.Plant tissue fed on by FAW was then scaled from 0-4 to analyze extent of herbivory on each individual plant 45 .Pre-exposure and post-exposure mass were then used to calculate mass gain normalized for initial mass 42 .This experiment was replicated on all three of the stated sorghum-sudangrass stages, 10-days old (3-leaf stage), 25-days old (panicle inflation stage), 60-days old (booting stage).

Continuous FAW exposure experiment with fresh leaves
For this experiment, FAW eggs were allowed to hatch inside the laboratory at 25 ℃ and were then reared exclusively on freshly cut sorghum-sudangrass leaves (leaves harvested for these bioassays were from young, fully developed from the top, ~10 leaves) from each of the six treatments: S425 (non-BMR) treated and untreated, S60 (BMR) treated and untreated, S72 (BMR) treated and untreated, N = 120.FAW were individually placed in plastic cups closed with lids (Dart Container Corporation, Mason, Michigan, USA) with enough plant material to never be starved.Cups were cleaned daily to avoid excess humidity and frass, alongside sorghum-sudangrass leaves replaced daily.Twenty caterpillars were reared on an artificial wheat-based germ diet, as described previously, to serve as control, N = 20.Once caterpillars reached 2nd instar, they were weighed daily for analysis of growth and development 46 .This experiment was also replicated on all three stages, 10-days old (3- In this experiment, FAW eggs were allowed to hatch inside the laboratory at 25 ℃ and were then reared on different artificial wheat-germ-based diets.Control diet was made following supplier specifications of 1000 mL water, 8 g agar powder, and 200 g Lepidopteran diet powder; however, plant-based diets were made with the addition of 10% plant material (plant material was selected from the top of the plant, taking multiple, large fully developed leaves), such as 20 g plant material: 180 g Lepidopteran diet powder, based on our previous studies 42 to test host plant toxicity under controlled conditions.For this experiment, freshly collected leaf material was finely ground using mortar-and-pestle, and then added to the diet just before cooling.FAW were then placed prior to hatching on plant-based diets from each of the 6 treatments, S425 (non-BMR) treated and untreated, S60 (BMR) treated and untreated, S72 (BMR) treated and untreated, and control, N = 140, in the similar plastic cups as mentioned before.Artificial diet was cut into ~1 cm 3 blocks and replaced every 2 days to avoid desiccation and excessive moisture build up in the cup.Once caterpillars reached 2nd instar, they were weighed daily for estimating growth and development.This experiment was also replicated with plant material from all three sorghum-sudangrass stages, 10-days old (3-leaf stage), 25-days old (Panicle Inflation Stage), 60-days old (Booting Stage).

Statistical analysis
For all the experiments, our statistical model had four factors: sorghum-sudangrass age (10d, 25d, 60d), sorghumsudangrass genotype (S425, S60, S72) seed treatment (untreated, treated), and BMR presence (No BMR, contains BMR).We pooled genotypes to narrow our scope to age, seed treatment, and BMR presence, and used Analysis of Variance for continuous variables (Total Mass Gain, Early Mass Gain, Late Mass Gain, Time to Pupation, Pupal Mass, and Adult Mass), and Ordinal logistic regression for discrete scale date (Plant Damage).Pairwise post hoc comparisons were carried out using Tukey's test.

Mass gain
Pre-and post-exposure caterpillar weights (g) were recorded and mass gain was then calculated expressed as a %.

Continuous fresh leaf exposure experiment
Total average mass gain Total average mass gain (%) was calculated from the mean of each caterpillar's daily growth.Mass gain analysis for the effect of BMR (ANOVA: P = 0.0088; Fig. 3a) on caterpillars fed on fresh leaves continuously, found that caterpillars fed on BMR plants (43.11 ± 8.00) gained significantly more mass than caterpillars on the artificial diet control (8.85 ± 2.79, P = 0.0065), yet for both there was no significant difference with mass gain on non-BMR plants (26.89 ± 3.88).Mass gain analysis for the effect of seed treatment (ANOVA: P = 0.0066, Fig. 3b) on caterpillars fed on fresh leaves found that caterpillars fed on untreated plants (45.5 ± 9.69) gained significantly more mass than caterpillars on artificial diet control (8.85 ± 2.79; P = 0.046), yet for both there was no significant difference with mass gain on Treated plants (30.18 ± 4.07).Mass gain analysis for the effect of plant age (ANOVA: P < 0.001, Fig. 3c) on caterpillars fed on fresh leaves found that caterpillars fed on 10-day old plants (57.63 ± 11.1) gained significantly more mass (ANOVA: P < 0.001, Fig. 3c) than those fed on 25 (20.86 ± 5.76; P = 0.0053), and 60-day old plants (14.06 ± 1.12; P < 0.0001).

Time to pupation
Time to pupation was calculated from how long it took caterpillars to pupate from their hatching date.Time to pupation (days) analysis for the effect of BMR presence (ANOVA: P < 0.0001, Fig. 4a) on pupation times found no significant difference in pupation times between caterpillars on BMR leaves (26.02 ± 0.31) and non-BMR leaves (25.4 ± 0.99), yet BMR (P < 0.0001) and non-BMR (P = 0.0003) both took significantly longer to pupate than the artificial diet control (21.72 ± 0.46).Time to pupation analysis for the effect of seed treatments (ANOVA: P < 0.0001, Fig. 4b) on pupation times found no significant difference in pupation times between caterpillars on treated leaves (26.1 ± 0.65) and untreated leaves (25.9 ± 0.34) yet treated (P < 0.0001) and untreated (P < 0.0001) both took significantly longer to pupate than the artificial diet control (21.72 ± 0.46).Time to pupation analysis for the effect of plant age (ANOVA: P < 0.0001, Fig. 4c) on pupation times found caterpillars fed on 10-day old leaves (25.3 ± 0.33; P = 0.0137) took significantly longer to pupate than caterpillars fed on 25-day leaves (23.2 ± 0.75; P = 0.0018), which took significantly longer to pupate than those fed on 60-day leaves (19.2 ± 0.98).

Pupal mass
Pupal mass was calculated from pupal mass after the first day of pupation.Pupal mass analysis for the effect of BMR presence (ANOVA: P = 0.0019, Fig. 5a) on pupal mass found that caterpillars that pupated from feeding on BMR leaves (0.154 ± 0.005) had significantly lower pupal mass than the artificial diet control (0.207 ± 0.013; P = 0.0012), although neither had a significant difference in pupal mass than caterpillars fed on non-BMR leaves (0.182 ± 0.012).Pupal mass analysis for the effect of seed treatment (ANOVA: P = 0.0063, Fig 5b) on pupal mass found no significant difference between caterpillars fed on treated leaves (0.161 ± 0.007) and untreated leaves (0.163 ± 0.007), yet the artificial diet control pupae (0.207 ± 0.013) were significantly heavier than both (P=0.0305;P=0.0123 respectively).Pupal analysis for the effect of plant age (ANOVA: P = 0.0026, Fig. 5c) on pupal mass found that caterpillars that pupated from feeding on 25-day old plants (0.214 ± 0.016) were significantly heavier than those fed 10-day old plants (0.175 ± 0.042; P = 0.0311), and 60-day old plants (0.130 ± 0.033; P = 0.0031).

Adult mass
Adult mass was calculated from eclosed pupae.Adult mass analysis for the effect of BMR presence (ANOVA: P = 0.0030, Fig. 6a) on adult mass found that FAW that had fed on BMR leaves (0.179 ± 0.051) were significantly lower in mass than those fed on non-BMR leaves (0.209 ± 0.024; P = 0.0044), and the artificial diet control (0.17 ± 0.012; P = 0.0152).Adult mass analysis for the effect of seed treatment (ANOVA: P = 0.4509, Fig. 6b) on adult mass found that there was no significant difference between adults that had fed on treated (0.148 ± 0.032) leaves, untreated (0.156 ± 0.019) leaves, and the artificial diet control (0.179 ± 0.012).Adult mass analysis for the effect of plant age (ANOVA: P = 0.9031, Fig. 6c) on adult mass found that there was no significant difference between adults that had fed on 10 (0.168 ± 0.013), 25 (0.170 ± 0.02), and 60-day old plants (0.156 ± 0.028).

Plant-based diet exposure experiment
Total average mass gain.Total average mass gain was calculated from the mean of each caterpillar's daily growth.Mass gain analysis for the effect of BMR presence (ANOVA: P = 0.3854, Fig. 7a) on caterpillars fed on different artificial leaf-diets found no significant differences in mass gain between BMR diets (17.17 ± 2.20), non-BMR diets (22.4 ± 3.57), and the artificial diet control (17.6 ± 2.28).Mass gain analysis for the effect of seed treatment (ANOVA: P = 0.3854, Fig. 7b) on caterpillars fed on seed treated artificial leaf-diets found no significant differences (ANOVA: P = 0.3854, Fig. 7b) in mass gain between treated diets (20.5 ± 0.272), untreated diets (17.1 ± 2.60), and the artificial diet control (17.6 ± 2.28).Mass gain analysis for the effect of plant age (ANOVA: P < 0.0001, Fig. 7c) on caterpillars fed on artificial leaf-diets from different aged plants found caterpillars gained significantly more mass (P < 0.0001) on 10-day old plants (40.5 ± 3.95) than 25-day old plants (14.2 ± 1.37), which were significantly heavier than those on 60-day old plants (1.12 ± 1.02; P = 0.0005).
Time to pupation.Time to pupation was calculated from the length of time required from hatching to pupation.Time to pupation analysis for the effect of BMR presence (ANOVA: P = 0.0134, Fig. 8a) on pupation times found no significant difference in pupation times between caterpillars on BMR leaf-diets (20.05 ± 0.202) and non-BMR leaf diets (20.2 ± 0.238), yet BMR (P = 0.0102) and non-BMR (P = 0.0471) both took significantly less time to pupate than the artificial diet control (21.29 ± 0.45).Time to pupation analysis for the effect of seed treatment (ANOVA: P = 0.0003, Fig. 8b) on pupation times found significantly faster pupation times (P = 0.0120) between caterpillars on untreated leaf-diets (19.7 ± 0.177) than treated leaf-diets (20.6 ± 0.265) and the artificial control diet (25.9 ± 0.34; P = 0.0007).Time to pupation analysis for the effect of plant age (ANOVA: P < 0.0001,  Pupal mass.Pupal mass was calculated from pupal mass after the first day of pupation.Pupal mass (g) analysis for the effect of BMR presence (ANOVA: P = 0.0358, Fig. 9a) on pupal mass found that caterpillars that pupated from feeding on BMR diets (0.183 ± 0.004) were significantly lower (P = 0.0311) in mass than those on non-BMR diets (0.204 ± 0.008), although neither were significantly different from the artificial diet control (0.196±0.10).Pupal mass analysis for the effect of seed treatment (ANOVA: P = 0.5938, Fig. 9b) on pupal mass found no significant difference in mass between caterpillars that pupated by feeding on treated leaf-diets (0.187 ± 0.007), untreated leaf-diets (0.194 ± 0.004), and the artificial diet control (0.196 ± 0.057).Pupal mass analysis for the effect of plant age (ANOVA: P= 0.4662, Fig. 9c) on pupal mass found no significant difference in mass between caterpillars that pupated on 10 (0.185 ± 0.006), 25 (0.194 ± 0.005), and 60-day old leaf-diets (0.196 ± 0.0102).

Discussion
In this study, we evaluated the effect of BMR, seed treatment, and plant age on the growth and development of FAW.We monitored BMR sorghum-sudangrass x FAW interactions through multiple lenses: 48-hour snapshot exposure, continuous leaf feeding, and life cycle on artificial diet fortified with fresh leaf extract.Our hypothesis that seed treatments would not have an effect on FAW growth and development, or on their ability to damage sorghum-sudangrass was supported, with no effects from seed treatment.However, we also hypothesized that older plants would be the most detrimental to FAW, yet while that was mostly supported, in the 48 h exposure snapshot experiment we saw the opposite.As for BMR, we hypothesized differential effects throughout every experiment; however, BMR had no effect on FAW in larval stages until the adult and pupal stages.The snapshot exposure experiments allowed us to understand the immediate response of FAW larvae to the seed treatment, to BMR, but also how plant age affects these larvae in the short term.When sensing herbivory, plants are able to respond by mobilizing local defenses to areas of herbivore feeding, or by releasing volatile organic compounds with the purpose of attracting natural enemies 47,48 .FAW, however, are highly mobile and have been shown to evade plant defenses by damaging a plant and moving on to another plant, or by feeding on different tissues of the same plant [49][50][51] .The snapshot exposure experiments give insight into how FAW are affected when spatially constrained and unable to perform movement-based evasion of plant defenses.Both continuous experiments exploring the entire FAW life cycle shed light on if FAW can overcome adverse effects in their diet later in their life cycle, or if detriments brought upon by our factors are insurmountable.While some studies have observed the effect of one of these factors, very few studies have taken them together and thoroughly monitored the FAW growth and development.
First, we found that seed treatments appeared to have almost no effect on FAW growth and development.These findings echo those of Muraro's group 22 which found no effect from seed treatments on FAW after 20 days of sowing, alongside those of Assefa 52 which found seed treatments as ineffective and inconclusive for FAW control in Ethiopia.These results are also in line with Thrash's group 23 , in which they found FAW survival to be the most affected by seed treatments.The seed treatment in our experiment, spinosad, acts on the ganglia and central nervous system (CNS) of target insect pests and disrupting acetylcholine pathways and receptors 53,54 .Spinosad is biologically derived from soil bacteria, Saccharopolyspora spinosa, and has been reported to be promising for integrated pest management because of its biological origin, low off-target effects 55 .Additionally, it is not known to share cross-resistance with more widely used insecticides 55 .However, the issue our study and more recent research has shown is that spinosad is not always effective, and almost always requires concurrent use of another insecticide to achieve efficient control, or a dramatic increase in dosage, as 200 ppm showed little effects while 1000-2000 ppm displayed high mortality in Lepidoptera 56,57 .While spinosad has been shown to be effective in controlled tests, Hertlein's group 57 also found that full sunlight and a large presence of organic matter may reduce spinosad's efficacy, which may explain why we saw no effect of the insecticide in our snapshot experiment performed in a greenhouse, regardless of the phonological stage of the plant.Regarding seed treatments, our results indicate that seed treatments other than spinosad should be further evaluated for FAW control, alongside novel pest management strategies.Plant age as a factor in FAW growth and development is highly important as it serves to develop action and economic thresholds 58,59 .Predicting pest infestations, and specifically FAW infestations, has been a prime concern for researchers and growers, and understanding what plant stages are most resistant or susceptible is key to developing management practices based around planting dates with expected pest infestation periods, and applying this data to changing pest patterns alongside global warming [60][61][62][63] .Our most conclusive results were found when monitoring the effect of plant age on FAW.We found that FAW exposed to 10-day old sorghumsudangrass plants for a short period, i.e.48 h exposure, gained less mass after this period, had the most herbivory damage, and gained the most mass in both continuous leaves and diet experiments; however, on continuous leaf diet they took the longest to pupate.This contradicts our other pupation time results as they were found to pupate the fastest on plant-based artificial diets.FAW on continuous leaf diets had the lowest growth on 60-day old plants, alongside losing significantly more mass in the final days of development leading to pupation on plant-based diets when compared to those feeding on 10-day old diets.One study 64 that analyzed sorghum waxes found that different stages in plant growth had different amounts of wax, and different chemical compositions of wax, both of which were highly variable until the grain heads became apparent.Although FAW did not pupate slower than those on 25-day old diets (the slowest pupation rate), they pupated significantly slower than those on 10-day old diets.While these adverse effects of 25-day old plants are not uncommon, they are usually seen in the reproductive stages of the plant, while in sorghum this is close to the transition point between vegetative and reproductive stages [65][66][67] .
A major driver of this study was the lack of a body of work on BMR as a host plant resistance trait, and therefore more work on BMR may shed light on these promising results and their mechanism(s).The effect of FAW on sorghum-sudangrass and sorghum has been well studied, especially with regard to FAW feeding leading to elevated levels of flavonoid compounds in sorghum-FAW interactions 48 .Flavonoids are crucial in providing host plant resistance 46,68 , and in sorghum, this being further supported by a sequential herbivory study by Kundu 69 in which FAW feeding first lead to drastic defense trait induction, measured by accumulation of total flavonoids.Sorghum flavonoids also have been shown to impose stress on another feeding guild insect, corn leaf aphids (Rhopalosiphum maidis), increasing the likelihood of alate development and reduction in colonization numbers 70 .Our knowledge gap therefore lies in understanding how BMR affects these defenses.Our results indicate that that when fed on BMR fresh leaves continuously, FAW had significantly lower pupal mass than the artificial diet control and a significantly lower adult mass than both the artificial diet control and non BMR plants.In the same vein, we found that when fed on plant-based artificial diets, FAW that fed on BMR diets also had significantly lower pupal mass than their non BMR counterparts.In Lepidoptera, pupal mass is a traditional indicator of adult traits, which translate into fitness, therefore reductions in pupal mass have been shown to hinder the overall fitness of the insect 71,72 .This data supports the idea that BMR does have an effect on herbivore performance and shows that further studies into the effect of BMR on pest insects should be expanded outside of FAW.Also, since this was consistent across ages of the plant and seed treatments, we predict that BMR is affecting FAW possibly through sorghum defense pathways.One possibility is through the modification of lignin biosynthesis in BMR mutants that leads to cascading effects in the plant's ability to respond to biotic factors, primarily herbivore pressure 73 .One such hypothesis is that the reduced lignin pathway leads to an increase in flavonoid synthesis, which are known for their defense properties 74 .While it is understood that there are tradeoffs due to BMR mutations within the plant, such as changes in metabolites, silencing, or overexpression of genes, further studies are required to understand the effect of BMR on surface defenses, primarily epicuticular waxes and trichomes, which has not been studied thoroughly 48,69,75 .
The adverse effects of BMR and later plant ages (25 and 60 days) on FAW growth and development found in this study also warrant deeper examination into their mechanism and modes of action.For example, plant growth stage being harmful to FAW in later stages is most likely due to altered wax concentrations 76,77 and secondary metabolites 78,79 found on older leaves.However, this does not explain the few cases where 25-day old leaves were more harmful to FAW growth and development than 60-day old leaves, although this could be tested by quantifying the wax content and abundance since only few studies have examined it 80 .
To conclude, our data from snapshot exposure, continuous fresh leaf exposure, and plant-based artificial diets-a collective and comprehensive examination of FAW growth and development, strongly supports the idea that BMR and plant age play a large role in deterring FAW herbivory by impeding their growth and development.Moving forward, the mechanisms of how these coalesce with plant resistance should be explored through studies that quantify differences in primary and secondary metabolites, and direct and indirect defenses, an area we are currently exploring.

Figure 1 .
Figure 1.Mean mass gain of 48 h snapshot exposure experiments for caterpillars on (a) Artificial diet control, non-BMR, BMR.(b) Artificial diet control, seed-treated, no seed treatment (c) 10-day old plants, 25 day old plants, and 60-day old plants.Each error bar is constructed using 1 standard error from the mean, different letters above the bars indicate significant differences among total mass gain between BMR treatments, seed treatments, and plant age determined by post hoc analyses using Tukey's test (P < 0.05).

Figure 2 .
Figure 2. Mean plant damage (0-4) of 48 h snapshot exposure experiments for caterpillars on (a) non-BMR and BMR.(b) Seed-treated and no seed treatment (c) 10-day old plants, 25-day old plants, and 60-day old plants.Each error bar is constructed using 1 standard error from the mean, different letters above the bars indicate significant differences among plant damage between BMR treatments, seed treatments, and plant age determined by post hoc analyses using Tukey's test (P < 0.05).

Figure 3 .
Figure 3. Mean total average mass gain of Continuous leaf exposure experiments for caterpillars on (a) Artificial diet control, non-BMR, BMR.(b) Artificial diet control, seed-treated, no seed treatment.(c) 10-day old plants, 25-day old plants, and 60-day old plants.Each error bar is constructed using 1 standard error from the mean, different letters above the bars indicate significant differences among total mass gain between BMR treatments, seed treatments, and plant age determined by post hoc analyses using Tukey's test (P < 0.05).

Figure 4 .
Figure 4. Mean time to pupation of continuous leaf exposure experiments for caterpillars on (a) Artificial diet control, non-BMR, BMR.(b) Artificial diet control, seed-treated, no seed treatment (c) 10-day old plants, 25-day old plants, and 60-day old plants.Each error bar is constructed using 1 standard error from the mean, different letters above the bars indicate significant differences among time to pupation between BMR treatments, seed treatments, and plant age determined by post hoc analyses using Tukey's test (P < 0.05).

Figure 5 .
Figure 5. Mean pupal mass of continuous leaf exposure experiments for caterpillars on (a) Artificial diet control, non-BMR, BMR.(b) Artificial diet control, seed-treated, no seed treatment.(c) 10-day old plants, 25-day old plants, and 60-day old plants.Each error bar is constructed using 1 standard error from the mean, different letters above the bars indicate significant differences among pupal mass between BMR treatments, seed treatments, and plant age determined by post hoc analyses using Tukey's test (P < 0.05).

Figure 6 .
Figure 6.Mean adult mass of Continuous leaf exposure experiments for caterpillars on (a) Artificial diet control, non-BMR, BMR.(b) Artificial diet control, seed -treated, no seed treatment.(c) 10-day old plants, 25-day old plants, and 60-day old plants.Each error bar is constructed using 1 standard error from the mean, different letters above the bars indicate significant differences among adult mass between BMR treatments, seed treatments, and plant age determined by post hoc analyses using Tukey's test (P < 0).

Figure 7 .
Figure 7. Mean total average mass gain of plant-based artificial diet experiments for caterpillars on (a) Artificial diet control, non-BMR, BMR.(b) Artificial diet control, seed treated, no seed treatment.(c) 10-day old plants, 25-day old plants, and 60-day old plants.Each error bar is constructed using 1 standard error from the mean, different letters above the bars indicate significant differences among total mass gain between BMR treatments, seed treatments, and plant age determined by post hoc analyses using Tukey's test (P < 0.05).

Figure 8 .
Figure 8. Mean time to pupation of plant-based artificial diet experiments for caterpillars on (a) Artificial diet control, non-BMR, BMR (b) Artificial diet control, seed-treated, no seed treatment (c) 10-day old plants, 25-day old plants, and 60-day old plants.Each error bar is constructed using 1 standard error from the mean, different letters above the bars indicate significant differences among time to pupation between BMR treatments, seed treatments, and plant age determined by post hoc analyses using Tukey's Test (P < 0.05).

Figure 9 .
Figure 9. Mean pupal mass of plant-based artificial diet experiments for caterpillars on (a) Artificial diet control, non-BMR, BMR.(b) Artificial diet control, seed-treated, seed treatment.(c) 10-day old plants, 25-day old plants, and 60-day old plants.Each error bar is constructed using 1 standard error from the mean, different letters above the bars indicate significant differences among pupal mass between BMR treatments, seed treatments, and plant age determined by post hoc analyses using Tukey's test (P < 0.05).

Figure 10 .
Figure 10.Mean adult mass of plant-based artificial diet experiments for caterpillars on (a) Artificial diet control, non-BMR, BMR.(b) Artificial diet control, seed treated, no seed treatment.(c) 10-day old plants, 25-day old plants, and 60-day old plants.Each error bar is constructed using 1 standard error from the mean, different letters above the bars indicated significant differences among adult mass between BMR treatments, seed treatments, and plant age determined by post hoc analyses using Tukey's test (P < 0.05).